The growth of normal cells is arrested when they come in contact with each other, a process known as contact inhibition. Contact inhibition is lost during tumorigenesis, resulting in uncontrolled cell growth. Here, we investigated the role of the tetraspanin transmembrane 4 superfamily member 5 (TM4SF5) in contact inhibition and tumorigenesis. We found that TM4SF5 was overexpressed in human hepatocarcinoma tissue. TM4SF5 expression in clinical samples and in human hepatocellular carcinoma cell lines correlated with enhanced p27 Kip1 expression and cytosolic stabilization as well as morphological elongation mediated by RhoA inactivation. These TM4SF5-mediated effects resulted in epithelial-mesenchymal transition (EMT) via loss of E-cadherin expression. The consequence of this was aberrant cell growth, as assessed by S-phase transition in confluent conditions, anchorage-independent growth, and tumor formation in nude mice. The TM4SF5-mediated effects were abolished by suppressing the expression of either TM4SF5 or cytosolic p27 Kip1 , as well as by reconstituting the expression of E-cadherin. Our observations have revealed a role for TM4SF5 in causing uncontrolled growth of human hepatocarcinoma cells through EMT.

Glycogen synthase kinase-3 (GSK-3) is a master regulator of growth and death in cardiac myocytes. GSK-3 is inactivated by hypertrophic stimuli through phosphorylation-dependent and -independent mechanisms. Inactivation of GSK-3 removes the negative constraint of GSK-3 on hypertrophy, thereby stimulating cardiac hypertrophy. N-terminal phosphorylation of the GSK-3 isoforms GSK-3␣ and GSK-3␤ by upstream kinases (e.g., Akt) is a major mechanism of GSK-3 inhibition. Nonetheless, its role in mediating cardiac hypertrophy and failure remains to be established. Here we evaluated the role of Serine(S)21 and S9 phosphorylation of GSK-3␣ and GSK-3␤ in the regulation of cardiac hypertrophy and function during pressure overload (PO), using GSK-3␣ S21A knock-in (␣KI) and GSK-3␤ S9A knock-in (␤KI) mice. Although inhibition of S9 phosphorylation during PO in the ␤KI mice attenuated hypertrophy and heart failure (HF), inhibition of S21 phosphorylation in the ␣KI mice unexpectedly promoted hypertrophy and HF. Inhibition of S21 phosphorylation in GSK-3␣, but not of S9 phosphorylation in GSK-3␤, caused phosphorylation and down-regulation of G1-cyclins, due to preferential localization of GSK-3␣ in the nucleus, and suppressed E2F and markers of cell proliferation, including phosphorylated histone H3, under PO, thereby contributing to decreases in the total number of myocytes in the heart. Restoration of the E2F activity by injection of adenovirus harboring cyclin D1 with a nuclear localization signal attenuated HF under PO in the ␣KI mice. Collectively, our results reveal that whereas S9 phosphorylation of GSK-3␤ mediates pathological hypertrophy, S21 phosphorylation of GSK-3␣ plays a compensatory role during PO, in part by alleviating the negative constraint on the cell cycle machinery in cardiac myocytes.cardiac hypertrophy ͉ heart failure ͉ mouse model ͉ signal transduction

SummaryChromatin is highly dynamic and subject to extensive remodeling under many physiologic conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We found increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1b) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver, we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In the skeletal muscle, we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogeneinduced cellular senescence, and are prominently found in terminally differentiated, postmitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to investigate further the progressive epigenetic changes that occur during aging.